Pepmixes to generate multiviral ctls with broad specificity

ABSTRACT

The present invention concerns methods of generating CTLs that are able to target at least one antigen from two or more viruses. The method includes exposing mixtures of peptides for different antigens to the same plurality of PBMCs and, at least in certain aspects, expanding the cells in the presence of IL4 and IL7.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. NonProvisional patentapplication Ser. No. 14/377,825 filed Aug. 8, 2014, which is a nationalphase application under 35 U.S.C. § 371 of International Application No.PCT/US13/25342 filed Feb. 8, 2013, which claims priority to U.S.Provisional Application Ser. No. 61/596,875 filed Feb. 9, 2012, all ofwhich are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grants U54HL081007 and N01-HB-10-03 awarded by National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The present invention generally concerns the fields of immunology, cellbiology, molecular biology, and medicine.

BACKGROUND OF THE INVENTION

Although hematopoietic stem cell transplant (HSCT) may curehematological malignancies and genetic disorders, extension to donorsother than HLA-matched siblings has resulted in the emergence of viralinfections as major contributors to post-transplant morbidity andmortality ¹⁻⁴. With the advent of more intensive viral screening andimproved detection, increasing numbers of viral pathogens have beenimplicated in these complications, expanding from cytomegalovirus (CMV),Epstein-Barr virus (EBV), herpes-simplex virus (HSV), Adenovirus (Adv),and BK to include human herpesvirus (HHV)-6, Respiratory Syncytial virus(RSV), parainfluenza, and influenza ². While pharmacological agents arestandard therapy for some, they have substantial toxicities, generateresistant variants, are frequently ineffective and do not providelong-term protection ^(5,6).

Restoration of virus-specific immunity offers an attractive alternativeto conventional drugs. The inventors have shown that in vitro expandedvirus-specific cytotoxic T lymphocytes (CTL) generated from stem celldonors with specificity for one (EBV), two (EBV and Adv) or three (EBV,CMV and Adv) viruses are safe and effectively prevent and treat viralinfection or disease in the HSCT setting ⁷⁻⁹. More recently, banked,partially HLA-matched virus-specific CTL (3rd party CTLs) are showingpromise in allograft recipients with advanced viral disease ¹⁰⁻¹².

Despite these encouraging clinical results broader implementation of Tcell therapy is restricted by (i) the limited spectrum of viruses thatcan be effectively targeted in a single T cell line, and (ii) thelogistics of manufacture. Antigenic competition between high and lowfrequency T cells as well as between multiple antigens expressed atdifferent levels and competing for presentation on shared antigenpresenting cells (APCs) may favor generation of lines dominated byresponses to a single virus or to a restricted spectrum of viralantigens ^(13,14), thus limiting the antiviral coverage provided by asingle T cell product. In addition, our current manufacturing process iscomplex, requiring infectious virus material (EBV/Adv), production of aclinical grade vector, and prolonged (10-12 weeks) in vitro culture^(8-10,15). To address this latter problem some groups have evaluatedmore rapid approaches for producing T cell products for adoptivetransfer. These include streptamer selection to directly isolatevirus-specific CD8+ T cells from peripheral blood ¹⁶, as well as theselection of cells based on cytokine production (IFN) or expression ofactivation markers (e.g. CD154) following antigen exposure ¹⁷⁻¹⁹.However, these approaches are expensive, require a large starting bloodvolume, which is not always available, particularly in the matchedunrelated donor setting, and cannot be applied to viruses with lowcirculating T cell precursor frequencies.

There is a need in the art for a mechanism by which one can rapidlygenerate a single preparation of polyclonal CTLs that is consistentlyspecific for immunodominant and/or subdominant antigens derived frommore than one virus, including those that are frequent causes ofpost-transplant disease or death, for example.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions thatconcern immune system components that are modified to immunogenicallyrecognize particular targets. In some embodiments, the present inventionconcerns the development of cytotoxic T-lymphocytes (CTLs) that target abiological moiety that elicits an immune response in an individual. Inspecific embodiments, the present invention concerns the development ofCTLs that target at least one antigen from a pathogen (including viral,bacterial, or fungal) or other disease-associated antigen. In certainaspects of the invention, the present invention concerns the developmentof CTLs that target antigens from at least one virus, for example. Inalternative embodiments, the present invention concerns the developmentof CTLs that target at least one tumor antigen, for example. In at leastsome cases, the CTLs target antigens from two or more viruses (or two ormore tumors, in alternative embodiments). In some embodiments, the CTLstarget one or more, two or more, three or more, or four or more antigensfrom the same virus. In some embodiments, the CTLs target one antigenfrom more than one virus. In certain embodiments, the CTLs target one ormore, two or more, three or more, or four or more antigens fromdifferent viruses.

The present invention provides significant and non-obvious improvementson methods for generating CTL lines with specificity against multipletumor antigens or multiple viruses (for example). In the generation ofCTLs with such specificity, the present invention obviates the need fordendritic cells in the preparation of such lines. In some cases, theantigen is presented to PBMCs in the form of one or more peptides thatspan some or all of the antigen. The antigenic peptides may be providedto the PBMCs in a library of peptide mixtures, which may be referred toas pepmixes. In other aspects of the invention, in the preparation ofthe CTLs the invention allows for the pooling of a variety of pepmixes.In some cases, the collection of antigens may include bothimmunodominant and subdominant antigens, yet despite the presence ofimmunodominant antigens in the collection with subdominant antigens,CTLs specific antigens including subdominant antigens are surprisinglygenerated.

In some embodiments of the invention, an individual is in need of themethods and/or compositions of the invention. In specific embodiments,the individual is immunocompromised (which for example, may be definedas an individual whose ability to fight infectious disease or cancerwith the immune system is compromised or entirely absent). In specificembodiments, the immunocompromised individual has had a stem celltransplant, has had an organ transplant and/or has received one or morecancer treatments, including chemotherapy or radiation, for example. Insome cases, the individual acquired or inherited immune deficiencydisorder. In some embodiments, those that are immunocompromised by theirdisease and/or its treatment are provided methods and/or compositions ifthe invention.

In some embodiments of the invention, there is a mechanism by which onecan rapidly generate a single preparation of polyclonal (for example,CD4+ and CD8+) CTLs that are consistently specific for a variety ofimmunodominant and/or subdominant antigens derived from one or moreviruses (for example, EBV, CMV, Adv, BK virus, HHV6, RSV and Influenza)that are frequent causes of post transplant disease or death. Theinvention is readily adaptable to clinical implementation and is usefulas an “off the shelf” broad spectrum antiviral agent. The invention usesstandardized (synthetic) peptides as a stimulus and enhancement ofcytokines to promote the survival and expansion of T cells, is readilyadaptable to clinical implementation, and is useful as a safe andeffective broad spectrum antiviral agent for all high risk transplantrecipients, for example.

In some embodiments of the invention, there is a method of generatingcytotoxic T-lymphocytes (CTLs) that target at least one antigen from twoor more viruses, comprising the steps of: contacting a plurality ofperipheral blood mononuclear cells with at least two libraries ofpeptides, said libraries of peptides each comprising peptides thatcorrespond to a particular viral antigen; and expanding the plurality ofcells in the presence of one or more cytokines. In specific embodiments,the method occurs in the absence of exposing the libraries to isolatedpeptide-pulsed dendritic cells prior to expanding the CTLs. In certainembodiments, the one or more cytokines are selected from the groupconsisting of IL4, IL7 and a combination thereof. In some embodiments,the peptides are further defined as peptides that overlap in sequence tospan part or all of a viral antigen. For example, in certain aspects thepeptides overlap by at least three, four, five, or six amino acids, andin some embodiments the peptides are at least six, seven, or eight ormore amino acids in length.

In some embodiments of the invention, there viruses targeted in theinvention are selected from the group consisting of EBV, CMV,Adenovirus, BK virus, HHV6, RSV, Influenza, Parainfluenza, Bocavirus,Coronavirus, LCMV, Mumps, Measles, Metapneumovirus, Parvovirus B,Rotavirus, West Nile Virus, JC, HHV7, and a combination thereof. Inspecific aspects, the virus is EBV and the antigen is selected from thegroup consisting of EBNA1, LMP2, and BZLF1. In specific aspects, thevirus is CMV and the antigen is selected from the group consisting ofIE1 and pp65. In specific cases, the virus is Adv and the antigen isselected from the group consisting of Hexon and penton. In someembodiments, the virus is BK virus and the antigen is selected from thegroup consisting of LT and VP-1. In some embodiments, the virus is HHV6and the antigen is selected from the group consisting of U14 and U90. Inspecific aspects, the virus is RSV and the antigen is selected from thegroup consisting of N and F. In certain embodiments, the virus isInfluenza and the antigen is selected from the group consisting of MP1and NP1.

In at least some methods of the invention, the CTLs generated therebyare administered to an individual, for example, an immunocompromisedindividual. In some cases, the individual has had allogeneic stem celltransplant. In specific embodiments, the cells are administered byinjection, such as intravenous, intramuscular, intradermal,subcutaneous, intraperitoneal injection, and so forth, for example. Insome embodiments, the individual has lymphoma or leukemia. In someembodiments, the CTLs are further defined as polyclonal CD4+ and CD8+CTLs. The PBMCs may be allogeneic to the individual or are autologous tothe individual. In some embodiments, the methods of the inventionfurther comprise the step of exposing the CTLs to one or morecompositions that stimulate cell division, such as phytohemagglutinin;in some aspects the compound is a mitogen.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIG. 1: Growth promoting cytokines enhance the activation and expansionof antigen-specific CTLs. PBMC were stimulated with pp65 pepmix in thepresence of IL2, IL15, IL4+7 or without exogenous cytokines. Cellexpansion were evaluated after 9-11 days of culture by cell countingusing trypan blue exclusion (n=5). Results are shown as mean cellnumbers+/−SEM. (A). Panel B CD3+ T cell proliferation in the differentculture conditions as evaluated by CFSE dilution. M1 shows thepercentage of cells that underwent at least 7 cell doublings on day 10after stimulation. Bulk cultures were analyzed for T and NK-cell markerexpression on day 10 after activation. Mean expression+/−SEM in CTLlines generated from 5 donors are shown in Panel C. Panel D showscytokine production from CD3/CD4+ (helper) and CD3/CD8+ (cytotoxic) CTLson day 9 after initiation in one representative donor (dot plots shownwere gated on CD3+ cells). Summary intracellular cytokine productionresults from three donors (mean+/−STDEV) are shown in Panel E. Finallythe cytokine production profile of pp65-specific CTL initiated with orwithout cytokines was evaluated by multiplex assay using supernatantharvested 18 h after antigenic restimulation (n=4). Th1 cytokines areshown in the left panel while prototypic Th2 cytokines are shown in theright panel (Panel F). Presence of regulatory T cells were evaluated byFoxP3 staining. Plots shown are gated on CD3+/CD4+ CTLs (Panel G).

FIG. 2: Peptide-stimulated and plasmid-activated CTLs share similarphenotypic and functional characteristics. Panel A CTLs were stimulatedeither directly with a pp65 pepmix or using DCs nucleofected with a DNAplasmid encoding the same antigen. Cell expansion was evaluated bycounting using trypan blue exclusion (n=4). Panel B shows the expressionof cell surface markers (average+/−STDEV expression) on CTLs 11 daysafter stimulation (n=4). The breadth of T cell reactivity in plasmid andpepmix-activated pp65-specific CTLs was evaluated by IFN ELIspot on day9 using a total of 22 mini peptide pools representing all pp65peptides.Data were normalized to 100% for maximum number of SFC per 1×10⁵ CTL.(Panel C). Panel D shows the TCR avidity of plasmid vs. pepmix activatedCTL generated from 2 representative donors. To assess avidity pp65-CTLswere stimulated with serial dilutions of pp65 pepmix (pp65) or relevant(HLA-matched) epitope peptides (NLV, QAD). IFN release of stimulatedCTLs was evaluated by ELIspot assay and maximum SFC/1×10⁵ cells wasnormalized to 100% for comparison purposes.

FIG. 3: Peptide length does not affect breadth of reactivity. Panel Ashows a schematic of three peptide libraries spanning a portion ofAdv-Hexon that were used for CTL initiation. Peptide libraries consistedof 15aa, 20aa or 30aa peptides covering the immunogenic C-terminal 414aaof Adv-Hexon. B Phenotypic analysis of CTLs performed on day 10 afterstimulation (n=6). Results are shown as mean+/−SEM. Breadth ofreactivity was tested using IFNγ ELIspot as a readout, with the 15 merHexon overlapping peptide library divided into mini-pools such that eachpool contained 5-6 contiguous peptides, as a stimulus.

FIG. 4: Pepmix-activated trivirus-specific CTL lines show similarspecificity to plasmid-activated T cells. CTL lines were generated usingDCs nucleofected with DNA plasmids encoding EBNA-1, LMP2, BZLF-1 (EBV),Hexon, Penton (Adenovirus), IE-1 and pp65 (CMV) or direct PBMCstimulation with the corresponding pepmixes. Specificity was determined10 days after initiation with IFN ELIspot as readout. Results areexpressed as SFC/1×10⁵ input cells. Control was IFN release in responseto stimulation with irrelevant pepmix.

FIG. 5: Generation of multivirus-specific CTLs. Panel A shows aschematic of antigen pooling strategy for CTL initiation. PBMCs werestimulated with pepmixes pooled by virus (A), divided intosub-pools—immunodominant and subdominant (B), divided into sub-poolsencompassing antigens from latent or lytic viruses (C), and finally allantigens were pooled together in a mastermix (D). After activation PBMCswere pooled and transferred to the G-Rex10 (15×10⁶/G-Rex). After 10 daysthe specificity of the CTL lines generated using these 4 poolingstrategies were analyzed using IFN ELIspot assay as readout andindividual pepmixes as a stimulus. Results from 2 representative donorsare presented in Panel B showing no difference in the specificity oflines. Panel C confirms that multivirus CTL can be reproduciblygenerated by pooling all pepmixes into one mastermix for activation(n=8). Results are expressed as SFC/1×10⁵ input cells+/−SEM. Control wasIFN release in response to stimulation with an irrelevant pepmix.Antigen specificity of CD3/CD8+ (cytotoxic) and CD3+CD8− (helper) Tcells was evaluated by intracellular IFN staining after overnightstimulation with the equivalent antigens. Results from onerepresentative donor are shown in Panel D. Panel E shows that the linesare polyfunctional as assessed using ICS for IFN and TNF in onerepresentative donor.

FIG. 6: Multivirus-specific CTLs can be expanded in vitro. On day 9after initial stimulation CTLs were restimulated using pepmix-pulsed PHAblasts. Panel A shows the expansion of CTLs from initiation (day 0) today 16, following a 2nd stimulation on day 9/10 (n=4). CTL expansion wasevaluated using trypan blue exclusion and results are shown as mean cellnumbers+/−STDEV. Panel B shows results from 1 representative donorillustrating the antigen specificity of CD3/CD8+ and CD3/CD8− (CD4+)CTLs after the 2nd round of stimulation using IFN ICS. Panel C showssummary results from 6 donors after the 1st (day 9) and 2nd (day 16)stimulation, using IFN ELIspot as a readout. Results are expressed asSFC/1×10⁵ input cells+/−STDEV and the control was IFN release inresponse to stimulation with irrelevant pepmix. The cytotoxic abilitiesof the generated CTLs were evaluated by standard 4-6 hr Cr⁵¹ releaseassay using pepmix-pulsed PHA blasts as targets. Specific lysis afterthe 1st and 2nd stimulation from 2 representative donors are shown inPanel D.

FIG. 7: Phenotype and specificity of Penton and LMP2-specific CTLsgenerated in the presence of different growth-promoting cytokines.Phenotypic analyses of CTLs on day 9 after initiation with Penton (upperleft) or LMP2 (upper right panel) pepmixes and culture in the presenceor absence of different cytokines. Results are presented as mean %positive cells+/−STDEV. CTLs of 3 donors were tested for specificity byIFN ELIspot. Results are expressed as SFC/1×10⁵ input cells+/−STDEV andthe control was IFN release in response to stimulation with anirrelevant pepmix.

FIG. 8: CD3+ T cell expansion after addition of growth-promotingcytokines. Total T cell numbers were calculated based on total cellnumbers evaluated by cell counting using trypan blue exclusion and thepercentage of CD3+ T cells detected on day 9 after CTL initiationassessed by flow cytometric analysis. Results from 5 donors are shown(mean cell numbers+/−SEM).

FIG. 9: TCR avidity is comparable in Hexon DC plasmid-activated andpepmix-stimulated PBMCs. TCR avidity of Hexon-specific CTLs stimulatedwith plasmid nucleofected DCs or pepmix stimulated PBMCs was tested byserial dilution of Hexon pepmix or HLA-A1 restricted peptide TDL withIFN ELIspot as readout. Results are plotted as % of maximum SFC.

FIG. 10: Comparable expansion of CTLs stimulated with pooled vs. singlepepmixes. Cell expansion of CTL generated from 2 donors was evaluatedusing trypan blue exclusion 9 days after PBMC stimulation. Results areexpressed as mean cell numbers+/−STDEV.

FIG. 11: Lack of alloreactivity in pepmix-stimulated PBMCs. Thealloreactive potential of pepmix-activated CTL stimulated either once(n=4) or twice (n=2) was tested by Cr⁵¹ release assay against a range ofallogeneic HLA-mismatched PHA blasts as targets.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The term “tumor antigen” as used herein refers to an antigenic substanceproduced/expressed on tumor cells and which triggers an immune responsein the host.

The term “viral antigen” as used herein refers to an antigen that isprotein in nature and is closely associated with the virus particle. Inspecific embodiments, a viral antigen is a coat proteins.

II. General Embodiments of the Invention

In certain aspects of the invention, the present invention concerns thedevelopment of CTLs that target one or more antigens from at least onevirus or at least one tumor antigen, for example. In some cases, theCTLs target one or more antigens from two or more viruses or two or moretumors.

The present invention concerns methods for generating CTL lines withspecificity against multiple tumor antigens or multiple viruses in atleast general embodiments. In methods of producing CTLs the antigen ispresented to PBMCs (for example) in the form of one or more peptidesthat span some or all of the antigen. The antigenic peptides may beprovided to the PBMCs in a library of peptide mixtures, which may bereferred to as pepmixes, and multiple libraries of pepmixes may beprovided to the same collection of PBMCs. In some embodiments, thecollection includes both immunodominant and subdominant antigens.

In some embodiments, the present invention is utilized in individualsafter hematopoietic stem cell transplantation (HSCT) Severe and fatalviral infections remain common after HSCT. Adoptive transfer ofcytotoxic T lymphocytes (CTLs) specific for EBV, CMV and Adenoviralantigens can treat infections that are impervious to conventionaltherapies, but broader implementation and extension to additionalviruses are limited by competition between virus-derived antigens andtime-consuming and laborious manufacturing procedures. The inventionprovides a system that rapidly generates a single preparation ofpolyclonal (CD4+ and CD8+) CTLs that is consistently specific for 15immunodominant and subdominant antigens derived from 7 viruses (EBV,CMV, Adv, BK, HHV6, RSV and Influenza) that commonly causepost-transplant morbidity and mortality. CTLs can be rapidly produced(10 days) by a single stimulation of donor PBMCs with a peptide mixturespanning the target antigens in the presence of the potent pro-survivalcytokines IL4 and IL7. This approach reduces the impact of antigeniccompetition with a consequent increase in the antigenic repertoire andfrequency of virus-specific T cells. The present invention can bereadily introduced into clinical practice and is a cost-effectivealternative to common anti-viral prophylactic agents for allogeneic HSCTrecipients.

III. Pathogens and Pathogenic Antigens

In some embodiments of the invention, the generated CTLs are provided toan individual that has or is at risk of having a pathogenic infection,including a viral, bacterial, or fungal infection. The individual may ormay not have a deficient immune system. In some cases, the individualhas a viral, bacterial, or fungal infection following organ or stem celltransplant (including hematopoietic stem cell transplantation), or hascancer or has been subjected to cancer treatment, for example. In somecases the individual has infection following an acquired immune systemdeficiency.

The infection in the individual may be of any kind, but in specificembodiments the infection is the result of one or more viruses. Thepathogenic virus may be of any kind, but in specific embodiments it isfrom one of the following families: Adenoviridae, Picornaviridae,Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae,Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus,Rhabdoviridae, or Togaviridae. In some embodiments, the virus producesantigens that are immunodominant or subdominant or produces both kinds.In specific cases, the virus is selected from the group consisting ofEBV, CMV, Adenovirus, BK virus, HHV6, RSV, Influenza, Parainfluenza,Bocavirus, Coronavirus, LCMV, Mumps, Measles, Metapneumovirus,Parvovirus B, Rotavirus, West Nile Virus, Spanish influenza, and acombination thereof.

In some aspects the infection is the result of a pathogenic bacteria,and the present invention is applicable to any type of pathogenicbacteria. Exemplary pathogenic bacteria include at least Mycobacteriumtuberculosis, Mycobacterium leprae, Clostridium botulinum, Bacillusanthracis, Yersinia pestis, Rickettsia prowazekii, Streptococcus,Pseudomonas, Shigella, Campylobacter, and Salmonella.

In some aspects the infection is the result of a pathogenic fungus, andthe present invention is applicable to any type of pathogenic fungus.Exemplary pathogenic fungi include at least Candida, Aspergillus,Cryptococcus, Histoplasma, Pneumocystis, or Stachybotrys.

IV. Tumor Antigens

In embodiments wherein multiTAA-specific CTL are employed for thetreatment and/or prevention of cancer, a variety of TAA may be targeted.Tumor antigens are substances produced in tumor cells that trigger animmune response in a host.

Exemplary tumor antigens include at least the following:carcinoembryonic antigen (CEA) for bowel cancers; CA-125 for ovariancancer; MUC-1 or epithelial tumor antigen (ETA) or CA15-3 for breastcancer; tyrosinase or melanoma-associated antigen (MAGE) for malignantmelanoma; and abnormal products of ras, p53 for a variety of types oftumors; alphafetoprotein for hepatoma, ovarian, or testicular cancer;beta subunit of hCG for men with testicular cancer; prostate specificantigen for prostate cancer; beta 2 microglobulin for multiple myelomand in some lymphomas; CA19-9 for colorectal, bile duct, and pancreaticcancer; chromogranin A for lung and prostate cancer; TA90 for melanoma,soft tissue sarcomas, and breast, colon, and lung cancer. Examples oftumor antigens are known in the art, for example in Cheever et al.,2009, which is incorporated by reference herein in its entirety.

Specific examples of tumor antigens include at least CEA, MHC, CTLA-4,gp100, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCR alpha, trp2, TCR,MUC1, cdr2, ras, 4-1BB, CT26, GITR, OX40, TGF-α. WT1, MUC1, LMP2, HPV E6E7, EGFRvIII, HER-2/neu, MAGE A3, p53 nonmutant, NY-ESO-1, PSMA, GD2,Melan A/MART1, Ras mutant, gp 100, p53 mutant, Proteinase3 (PR1),bcr-ab1, Tyrosinase, Survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP,EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, Androgenreceptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, FucosylGM1, Mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn,GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX,PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1,B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2,and Fos-related antigen 1, for example.

V. Generation of Pepmix Libraries

In some embodiments of the invention, a library of peptides is providedto PBMCs ultimately to generate CTLs. The library in particular casescomprises a mixture of peptides (“pepmixes”) that span part or all ofthe same antigen. Pepmixes utilized in the invention may be fromcommercially available peptide libraries made up of peptides that are 15amino acids long and overlapping one another by 11 amino acids, incertain aspects. In some cases, they may be generated synthetically.Examples include those from JPT Technologies (Springfield, Va.) orMiltenyi Biotec (Auburn, Calif.). In particular embodiments, thepeptides are at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 ormore amino acids in length, for example, and in specific embodimentsthere is overlap of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, or 34 amino acids in length, for example. The mixture of differentpeptides may include any ratio of the different peptides, although insome embodiments each particular peptide is present at substantially thesame numbers in the mixture as another particular peptide.

VI. Combination Therapy

In certain embodiments of the invention that concern CTLs generatedagainst tumor antigens, methods of the present invention for clinicalaspects are combined with other agents effective in the treatment ofhyperproliferative disease, such as anti-cancer agents. An “anti-cancer”agent is capable of negatively affecting cancer in a subject, forexample, by killing cancer cells, inducing apoptosis in cancer cells,reducing the growth rate of cancer cells, reducing the incidence ornumber of metastases, reducing tumor size, inhibiting tumor growth,reducing the blood supply to a tumor or cancer cells, promoting animmune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cancer cells withthe expression construct and the agent(s) or multiple factor(s) at thesame time. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent(s).

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with gene therapy. For example, the herpessimplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors bya retroviral vector system, successfully induced susceptibility to theantiviral agent ganciclovir⁵¹. In the context of the present invention,it is contemplated that cell therapy could be used similarly inconjunction with chemotherapeutic, radiotherapeutic, orimmunotherapeutic intervention, in addition to other pro-apoptotic orcell cycle regulating agents.

Alternatively, the present inventive therapy may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and present invention are appliedseparately to the individual, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and inventive therapy would still be ableto exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4,5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed, present invention is “A” and thesecondary agent, such as radio- or chemotherapy, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/BB/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/AB/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

It is expected that the treatment cycles would be repeated as necessary.It also is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the inventivecell therapy.

A. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, abraxane, altretamine, docetaxel, herceptin,methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein tansferase inhibitors,transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate,or any analog or derivative variant of the foregoing.

B. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

C. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Immunotherapy, thus, could be used as part of a combined therapy, inconjunction with the present cell therapy. The general approach forcombined therapy is discussed below. Generally, the tumor cell must bearsome marker that is amenable to targeting, i.e., is not present on themajority of other cells. Many tumor markers exist and any of these maybe suitable for targeting in the context of the present invention.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155.

D. Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as the present invention clinical embodiments. A varietyof expression products are encompassed within the invention, includinginducers of cellular proliferation, inhibitors of cellularproliferation, or regulators of programmed cell death.

E. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs'surgery). It is further contemplated that the present invention may beused in conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

F. Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion, oragents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers. Immunomodulatory agents include tumor necrosisfactor; interferon alpha, beta, and gamma; IL-2 and other cytokines;F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, andother chemokines. It is further contemplated that the upregulation ofcell surface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5/TRAIL would potentiate the apoptotic inducing abililties of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increases intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyerproliferative efficacy of the treatments. Inhibitors ofcell adhesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

DNA methyltransferase inhibitors and/or histone deacetylase inhibitors.Exemplary DNA methyltransferase inhibitors include, for example,5-azacytidine, 5-aza-2′-deoxycytidine,1-beta-D-arabinofuranosyl-5-azacytosine and dihydro-5-azacytidine.Exemplary HDAC inhibitors include hydroxamic acids, such as trichostatinA; cyclic tetrapeptides (such as trapoxin B), and the depsipeptides;benzamides; electrophilic ketones; and the aliphatic acid compounds suchas phenylbutyrate and valproic acid.

VII. Kits of the Invention

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a library of pepmixes may be comprised in a kit,any type of cells may be provided in the kit, and/or reagents formanipulation of pepmixes and/or cells may be provided in the kit. Thecomponents are provided in suitable container means.

The kits may comprise a suitably aliquoted compositions of the presentinvention. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the components in closeconfinement for commercial sale. Such containers may include injectionor blow-molded plastic containers into which the desired vials areretained.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

EXAMPLES

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way,however, be construed as limiting the broad scope of the invention.

Example 1 IL2, IL15 and IL4+7 Promote the Expansion of Peptide-ActivatedT Cells In Vitro

To increase the range of viral antigens that could be recognized by asingle CTL line and to mitigate the impact of antigenic competition inorder to retain both high and low frequency T cells, the inventorsstimulated PBMCs in the presence of different Th1, pro-proliferative andpro-survival cytokines. The inventors then compared the frequency andrepertoire of responding cells to those generated by conventionalactivation in the absence of cytokines. In exploratory experiments PBMCswere simulated with a pepmix (peptide library of overlapping 15 mers)spanning the immunodominant CMV-pp65 antigen, then expanded withoutcytokines, or with media supplemented with (i) IL15 (5 ng/ml), (ii) IL2(20 U/ml), or (iii) IL4 (1666 U/ml)+IL7 (10 ng/ml). After 9-12 days theinventors assessed cell expansion, phenotype, specificity and function.

Cultures supplemented with IL15 or IL4+7 showed the greatest overallexpansion (5±0.6 and 3.7±0.5 fold increase, respectively) over 9 days(n=5). Cultures that were stimulated in the absence of cytokines did notexpand (0.6±0.04), while the IL2 condition was intermediate (2.7±0.1)(FIG. 1A). To determine whether the superior cell numbers were aconsequence of improved T cell proliferation, enhanced survival, or thecombination, cells were labeled with CFSE on day 0 and then analyzedevery 2-3 days to measure cell doubling, while live andapoptotic/necrotic cells were distinguished by Annexin-PI staining (notshown). Flow cytometric analysis demonstrated no difference in thenumber of cell divisions from day 0-5. However, from day 5 onward cellscultured in cytokines continued to divide, whereas in their absence,cell division was reduced and viability was consistently lower (FIG.1B). These data indicate that the improved survival of proliferatingcells made the primary contribution to the observed increase in cellnumbers in cytokine-supplemented cultures.

Example 2 IL4+7 Support the Selective Expansion of Polyclonal,TH1-Polarized T Cells

Optimal in vivo T cell persistence and activity requires both helper(CD4+) and cytotoxic (CD8+) T cells ²³. The inventors therefore usedphenotypic analyses to determine that the cells in thecytokine-supplemented cultures reflected the selective expansion ofpolyclonal T cells. The inventors found the lowest frequency of CD3+ Tcells in cultures supplemented with IL2 or IL15 (72.8±2.1% and61.3±3.7%, respectively), which instead contained significantly highernumbers of CD56+ NK cells than other conditions (27.1±2.3% and37.7±3.7%, respectively) (n=5). By contrast, IL4+7 cultures werecomprised almost entirely of CD3+ T cells (92.6±0.4%), with both CD8+ Tcells and significantly more CD4+ T cells (61±2.7%) than the othercytokine-supplemented conditions (IL2 26±4%, IL15 17.6±4.3%, p=0.024,p=0.004, respectively) (FIG. 1C). To confirm that both CD8+ and CD4+ Tcells were antigen-specific and produced effector cytokines theinventors performed intracellular cytokine staining (ICS) for IFN. FIG.1D shows representative results from 1 donor, while FIG. 1E showssummary results for 3 donors. The data confirm that IL4+7-supplementedcultures contained antigen-specific IFN-producing T cells in bothcompartments (CD4+ 39.3%±16.4%, CD8+ 22.2%±2.2%), at levelssubstantially higher than in other conditions (no cytokine: CD4+2.3%±3.9%, CD8+ 0.8%±0.5%; IL15: CD4+ 1.7%±1.4%, CD8+ 13.9%±2.8% andIL2: CD4+ 2.2%±3.1%, CD8+ 12.6%±2.6%, n=3). Similar results wereobtained using pepmixes from subdominant Adv (Penton) and EBV (LMP2)viral antigens; indeed outgrowth of NK cells was even more evident inthe IL2 and IL15-supplemented conditions (FIG. 7).

IL4 is a prototypic Th2 cytokine, therefore to more comprehensivelyevaluate the cytokine profile of the induced CTLs the supernatant ofantigen-activated T cells was assessed using luminex array. FIG. 1Fshows that, in addition to IFNγ, the IL4+7-supplemented lines producedthe prototypic Th1 cytokines GM-CSF, IL-2 and TNFα, at levels similar tothat of IL2-induced CTLs. In addition, levels of Th2 cytokines (IL5 andIL13) were not substantially different and there was no evidence ofregulatory T cell outgrowth, as assessed by CD4/CD25/FoxP3+ staining(FIG. 1G). Thus, IL4, in combination with IL7, induces selectiveexpansion of polyclonal, Th1-polarized T cells that produce multipleeffector cytokines upon stimulation (FIG. 8).

Example 3 Overlapping 15 Mer Peptide Libraries Activate T Cells withSimilar Specificity and Avidity to those Generated usingEndogenously-Processed Full Length Antigen

To address concerns that pepmixes might reactivate low avidity T cellsunable to recognize antigens that are naturally processed and presentedby virus-infected cells, the inventors compared pp65 pepmix-activatedCTLs with those generated using DCs nucleofected with a DNA plasmidencoding the same antigen ^(20,21). After activation, each set of cellswas expanded in IL4+7. Expansion was similar between the groups, with107±23.4×10⁶ cells generated using pepmix-pulsed PBMCs (7.2 foldexpansion) versus 130.3±46.9×10⁶ cells in the DC-stimulated cultures(8.7 fold expansion) (FIG. 2A) (n=3). Phenotypic analysis demonstratedthat the pepmix-activated CTLs were predominantly CD4+ (74.3±19.3%),with a minor CD8+ component (22.8±19.2%), as were the plasmid-activatedCTLs (CD4+ 70.6±14.2% and CD8+ 26.5±13.4%) and both expressed similarlevels of the memory and activation markers CD62L, CD28 and CD45RO(61±46.7%, 86.5±3.5%, 92±7.1% pepmix vs. 77±28.3%, 85.5±0.7%, 87.5±13.4%plasmid) (FIG. 2B). The inventors next compared the breadth of epitopesrecognized by measuring responses to 110 20 mer peptides (overlapping by15aa) spanning CMV-pp65 and arranged into 22 pools such that eachpeptide was represented in 2 pools ²⁴. FIG. 2C shows that both therecognition of a given peptide and the magnitude of the response theretowas little changed by the antigen source. Finally, the inventorscompared functional avidity by IFNγ ELIspot using log dilutions of thepp65 pepmix or epitope peptides (A2-NLV and A24-QAD) as a stimulus. Asshown in FIG. 2D, there was no significant difference in the avidity ofthe CTLs. This data was confirmed for other viral antigens usingAdv-Hexon pepmix and viral antigen-encoding plasmid as a stimulus (FIG.9).

Example 4 15 Mer Peptides Activate CD4+ and CD8+ T Cells as Efficientlyas Long (20 Mer or 30 Mer) Peptides

Since CD4+ epitopes (>20aa) may be longer that CD8 epitopes (8-10aa) theinventors next determined whether longer peptides would induce higherfrequencies of antigen-specific CD4+ T cells. The inventors obtainedthree overlapping peptide libraries (#1-15 mers overlapping by 11, #2-20mers overlapping by 15, and #3-30 mers overlapping by 15) spanning the Cterminus (aa539-953) of Adv-Hexon; a region rich in both CD4+ and CD8+epitopes ^(25,26) (FIG. 3A). The inventors directly stimulated PBMCswith each of the libraries and evaluated the phenotype, epitopespecificity and breadth of the lines.

Phenotypically the lines were comparable, with a predominance of CD4+cells (mean 56±5.5% vs. 59±5.8% vs. 60±6%) and a minor CD8+ component(mean 21±0.2% vs. 20±0.1% vs. 16±0.2%), and similar levels of the memoryand activation markers CD62L, CD28 and CD45RO (CD62L—60±1.9% vs. 57±1.9%vs. 51+/−1.6%, CD28—88±0.6% vs. 84±2.1%, vs. 89±0.6% and CD45RO—58±1.7%vs. 60±1.6% vs. 60±1.2%) (15 mer vs. 20 mer vs. 30 mer) (n=6). To learnwhether the spectrum of epitopes recognized differed based on thestimulating library; the inventors rechallenged the induced CTLs withsubpools of peptides from each library and found no consistent orstatistically significant differences in the breadth of peptidesrecognized. Results for the 15 mer minipool rechallenge are shown inFIG. 3C. Since 15 mer pepmixes are readily available as both researchand clinical products the inventors performed all subsequent experimentswith this antigen source.

Example 5 Generation of a Single T Cell Culture with SimultaneousSpecificity for ADV, EBV and CMV

After successfully generating CTLs using peptides derived from a singleviral antigen and culture in IL4+7, the inventors next prepared a singleculture of CTLs simultaneously recognizing CMV, EBV, and Adv. For eachvirus the inventors targeted immunogenic antigens; CMV—IE1 and pp65,Adv—Hexon and Penton, and EBV—EBNA1, LMP2 and BZLF1 ^(8,9,17,18,27-31)and pulsed PBMCs with the relevant pepmixes before culture in IL4+7.After 9-12 days the inventors compared the antiviral reactivity of theresulting CTLs with those generated using our current clinical trivirusCTL protocol which uses DCs nucleofected with plasmids encoding the sameantigens as a stimulus ^(20,21) (FIG. 4). IFNγ ELIspot confirmed thatpepmix-generated CTLs from 4 donors had antiviral activity against allthree viruses and seven stimulating antigens. The frequency of T cellsreactive against EBV (EBNA1, LMP2, BZLF1) and CMV (IE1, pp65) wascomparable irrespective of the stimulus. In contrast, all 4 donors hadsignificantly more Adv-reactive T cells (Hexon and Penton) inpepmix-stimulated cultures [Hexon—median 462.3, range 373-572.5 vs.median 112, range 53-421.5 SFC/2×105 CTL; p=0.01, Penton—median 317,range 105.5-345 vs. median 51.25, range 4-134 SFC/2×105 CTL, p=0.02,pepmix vs. plasmid, respectively].

Example 6 Extension to Additional Viruses

To determine whether the direct pepmix stimulation approach could beextended to generate multivirus-specific CTL lines targeting a broaderspectrum of different clinically relevant viruses the inventorsstimulated PBMCs with pepmixes spanning 2 or 3 T cell immunogenicantigens from CMV, Adv, EBV, BK, Influenza, RSV and HHV6 (Table 1).

TABLE 1 Exemplary Antigens from Exemplary Viruses Virus Antigen EBVEBNA-1, LMP2, BZLF1 CMV IE-1, pp65 Adenovirus Hexon, Penton BK virus LT,VP-1 Influenza MP1, NP1 RSV N, F HHV-6 U14, U90

To determine whether antigenic competition would preclude pooling theinventors segregated the pepmixes and stimulated PBMCs with minipoolscontaining pepmixes from, A) each virus; B) immunodominant (CMV, RSV,Flu, HHV6) and sub-dominant (Adv, EBV, BK) viruses; C) lytic (Adv, RSV,Flu) and latent (EBV, CMV, HHV6, BK) viruses, or D) a mastermix of allpepmixes (FIG. 5A). There was no difference in either the rate ofexpansion (FIG. 10), the overall specificity or magnitude of theresponse directed against each antigen, irrespective of the compositionof the stimulating pepmix pool (FIG. 5B). Thus, all further studies usedthe mastermix (condition D). FIG. 5C shows 8 additional CTL lines withconsistent multivirus specificity. The highest responses were seenagainst CMV-pp65 and Adv-Hexon (951.6±82.1 and 461.4±19.2 SFC/1×10⁵ CTL)while activity against HHV6-U90, EBV-BZLF1 and EBV-LMP2 was weakest(26.9±4.2, 35.6±5, 39.6±2.6 SFC/1×10⁵ CTL). Adv-Penton, Influenza-MP1and RSV-F demonstrated intermediate response rates (191±13.7, 117.6±8.6,90.1±10.3 SFC/1×10⁵ CTL, respectively) (FIG. 5C). The lines werepolyclonal and polyfunctional with activity against the stimulatingviruses detectable in both CD4+ and CD8+ fractions (FIG. 5D), andreactive cells produced both IFNγ and TNFα superior in vivoactivity^(32,33). FIG. 5E shows the results for one representative donorin whom 63% of all Adv, 55% of CMV, 40% of EBV, 46% of RSV, 36% ofInfluenza and 28% of HHV6-specific CTLs produced both IFNγ and TNFαafter antigenic stimulation. ICS for IFNγ and/or TNFα showed that67.7±13.3% of all T cells in multivirus cultures were antigen-specific.This percentage is likely an underestimate since some virus-specificCTLs do not produce cytokines or produce effector cytokines other thanIFNγ and TNFα ³³. Finally, even though these CTLs had received only asingle stimulation there was no evidence of alloreactivity, assessed byCr⁵¹ release assay using HLA-mismatched PHA blasts as targets (FIG. 10),an important consideration if these cells are to be used for thetreatment of allogeneic HSCT recipients.

Example 7 Multivirus-Specific CTL can be Expanded In Vitro

To discover whether multivirus-specific CTLs could be further expandedto provide numbers suited for third party or “off-the-shelf” use, theinventors restimulated the cells with autologous PHA blasts pulsed withthe same mastermix of pepmixes. Secondary expansion of a mean of 8.4±2fold was obtained over 7 days, to a final cell number of 604.6±23.7×106(FIG. 6A). FIG. 6B shows that the expanded CTLs remained polyclonal,with activity detected in both CD4+ and CD8+ compartments. Expansion wasassociated with an overall increase in the magnitude of the responsedirected against all of the stimulating antigens on day 16 relative today 9 (FIG. 6C) so that >80% of cells in the restimulated culturesproduced IFNγ and/or TNFα Similarly, these expanded cultures had greatercytolytic activity, ranging from >60% (CMV) to 14% (BK), demonstratingretained specificity for both sub-dominant and immunodominantantigens/viruses without alloreactivity (FIG. 11).

Example 8 Significance of Certain Embodiments of the Invention

The inventors have shown that in at least some embodiments they canrapidly generate polyclonal, CD4+ and CD8+ T cells with specificitiesdirected to a wide range of lytic and latent viruses responsible forinfection in the immunocompromised host and after HSCT. These cells wereTh1-polarized, had high avidity for a multiplicity of individual viralantigens, produced multiple effector cytokines upon stimulation, andkilled virus-infected targets without alloreactivity. Because theinventors generated these T cells using combinations ofclinically-available peptide-libraries and pro-survival cytokines, ourapproach should be well suited to clinical application.

While CMV, EBV and Adv are the most frequently detected viral infectionsfollowing allogeneic HSCT, recipients are also susceptible to numerousother viruses, including BK, JC, HHV6, HHV7, influenza, parainfluenza,coronavirus, and RSV, all of which may cause severe morbidity andmortality ^(1,2). Several of these viruses are only seasonally detected(e.g. influenza, RSV) while others, such as HHV7, JC, and coronavirus,are infrequent, so that it is impracticable to cover all these pathogenspost-transplant by generating individualized patient and singlevirus-specific T cell products. Hence, the inventors sought to develop astrategy that would enable the production of a single CTL line withsimultaneous specificity for a multiplicity of antigens.

In the current clinical trials of virus-specific T cells, the inventorshave used EBV-LCL, adenovectors and/or viral antigen-encoding DNAplasmids to generate virus-directed T cells ^(7-9,20,21). The use offull-length antigen ensures that CTL can be generated from all donors,irrespective of HLA, and that the antigen is physiologically processedby APCs and produces CTLs that recognize multiple CD4+ and CD8+ T cellepitopes and have sufficient avidity to kill virus-infected targets. Theinduction of lines that recognize multiple epitopes also minimizes virusescape due to epitope loss and produces potent and sustained anti-viralactivity in vivo ³⁴. However, the requirements for live virus/vectorsare barriers to broader and late phase clinical studies, and also limitthe number of pathogens to which a single T cell line can be directed^(8,9). The inventors therefore evaluated whether clinically applicablepepmixes could be used as an alternative. Though clinical studies usingminimal epitope peptides as vaccines have resulted in immune toleranceor the activation of low avidity T cells ³⁵, Melief and colleaguesrecently demonstrated improved results with long (22-45aa) peptidescontaining both CD4+ and CD8+ epitope sequences ³⁶. They observed thatthese long peptides were processed endogenously, presented to T cells byAPCs, and induced both helper and cytotoxic T cells, resulting in robustand effective CTL responses ³⁶. Based on these data, the inventors choseto use a whole antigen source in the form of overlapping peptidelibraries, but for optimal induction of polyclonal CTL the inventorscompared peptides of different lengths (15 mers, 20 mers and 30 mers)for stimulation. However, the inventors saw no difference in thephenotype, specificity or epitope breadth of our lines, highlighting thedifferences between delivering peptides as a vaccine, where one relieson endogenous APCs to take up and process antigen versus in vitro T cellactivation using professional APCs within PBMCs at optimaleffector:target ratios ³⁷. Given the ready clinical availability ofpepmixes containing 15 mer peptides that cover all possible CD8+ and themajority of CD4+ epitopes, the inventors substituted this antigen sourceand were able to demonstrate equivalency to “conventionally generated”CTLs with respect to both epitope specificity and avidity ^(20,21).

The inventors next addressed how best to extend the breadth ofantigen/epitope specificities that could be accommodated within a singleCTL line. Physiologically, T cells are activated when they receivesignals from TCR stimulation (signal 1), co-stimulation (signal 2), andcytokines (signal 3). The “conventional CTLs” are activated in theabsence of exogenous cytokines, a deficit that appears to adverselyaffect their proliferative capacity in vitro and also increases theirsusceptibility to activation induced cell death (AICD), likely resultingin a more restricted repertoire of epitope recognition. Consistent withthis possibility, both the frequency and breadth of cells with viralspecificity could be increased by supplementing cultures withinflammatory and pro-survival cytokines at initiation. The inventorschose to test cytokines that support cell proliferation in vitro and invivo (IL2, IL15) ^(38,39), as well as combinations (IL4+7) that alsosupport the retention of a central memory phenotype, and promote thesurvival of activated T cells by upregulation of anti-apoptoticmolecules e.g. Bcl-2 ⁴⁰⁻⁴³. Only lines supplemented with IL4+7selectively promoted the expansion and survival of both CD4+ and CD8+virus-specific T cells: of note, the induced cells were Th1-polarizeddespite exposure to IL4, a prototypic Th2 cytokine. Given the clinicalavailability of both cytokines and their safety in human clinical trials^(44,45), IL4+7 fulfilled the requirements of the current study, howeverother pro-inflammatory cytokines capable of mimicking the milieu presentduring viral infection may produce similar benefits. For example, vonRossum and colleagues recently reported that CD3/28-activated CD8+ Tcells cultured in an inflammatory cocktail consisting of IL1+IL6+IL23underwent significantly less cell death after activation as comparedwith cells activated in any of the cytokines alone or activated in thepresence of IL12 ⁴⁶.

The direct stimulation of PBMCs with pepmixes and culture incytokine-supplemented conditions also allowed us to overcome a secondmajor barrier to increasing the spectrum of viruses targeted in a singleCTL line, namely antigenic competition resulting from the use of acommon APC to simultaneously present multiple antigenic components fromdifferent viruses ^(8,9). Antigenic competition results both fromlimited access of peptides to HLA molecules and physical constraints onthe simultaneous stimulation of both high and low frequency T cells^(13,14). To overcome these issues, investigators have used artificialAPCs frequency T cells (AAPCs) that are engineered with molecules toprovide the necessary TCR and co-stimulatory events required for immunesynapse formation ⁴⁷. However, to avoid the inevitable complexities andcosts of introducing a gene-modified cellular product into themanufacturing process, the inventors evaluated whether patient PBMCsthemselves could act as both a source of antigen presenting andresponding cells. B cells, monocytes and macrophages may all have thecapacity to present antigen to T cells and these APCs can utilize endo-and exopeptidases to liberate class I or class II epitopes from 15 merpeptides ^(48,49). By taking advantage of these properties, theinventors can avoid reliance on a single APC endogenously expressingmultiple antigens at different levels as a shared T cell stimulator, andinstead have a diverse group of APCs in which each cell has thepotential to display a diverse repertoire of peptides, allowingsufficient access for both high and low frequency T cells. Thus,antigenic competition both within the APC and between T cells could bealleviated. As proof of principle, the inventors generated a singleculture of T cells with reactivity for 15 antigens derived from 7 latentand lytic viruses (EBV, CMV, BK, HHV6, Adv, Flu, and RSV) using pooledpepmixes as a stimulus and saw no evidence of competition. Additionalpathogens can be included in this platform, although in some embodimentsultimately APC numbers can eventually become limiting; thus additionsmust be performed in a stepwise manner and one must evaluate changes inthe frequency and breadth of T cell recognition of all peptides in themix.

Critically for clinical feasibility, the approach was able to producelarge numbers of virus-specific T cells. By seeding just 1.5×10⁷ PBMCsin the G-Rex and a single in vitro stimulation the inventors couldregularly manufacture 1×10⁸ CTLs within 10 days, with a >10-foldenrichment in virus-specific cells and a corresponding reduction inalloreactive T cells to levels observed in repetitively stimulatedconventional CTLs, which have a proven safety record in vivo ^(7-9,50).Thus, using our new manufacturing technology the inventors predict thatmultivirus-specific CTL will be safe for infusion after a singleexposure to pepmixes and will provide broad spectrum anti-viralprotection without GvHD. Should additional cells be required, forexample if banked virus-specific CTLs are established for 3rd partyrecipients, a second stimulation using pepmix-pulsed PHA blasts canexpand the total number of CTLs without impairing their epitopespecificity or breadth.

Example 8 Exemplary Materials and Methods

A. Donors and Cell Lines

PBMCs were obtained from healthy volunteers with informed consent usinga Baylor College of Medicine IRB-approved protocol. PBMCs were used togenerate DCs, CTL lines and PHA blasts. PHA blasts were generated fromPBMC (2×10⁶/ml) using PHA (5 μg/ml) and maintained in CTL media (RPMI1640, 45% Click's (Irvine Scientific, Santa Ana, Calif.), 2 mM GlutaMAXTM-I, and 5% Human AB Serum) supplemented with IL2 (100 U/ml, NIH,Bethesda, Va.), which was replenished every 3 days.

B. CTL Generation—Peptide Stimulation

i. Peptides/Pepmixes

For PBMC stimulation the inventors used commercially available pepmixes(15 mers overlapping by 11aa spanning EBV-LMP2, BZLF1, EBNA1;Adv-Penton, Hexon; CMV-pp65, IE-1; BKV-VP1, large T; Influenza A-MP1(H3N2), NP (H3N2); RSV-F, N, JPT Technology, Berlin, Germany. Pepmixesspanning HHV6 U14 and U90 were synthesized by Genemed Synthesis Inc.,San Antonio, Tex. USA. Peptide libraries spanning the 414aa C-terminusof Adv-Hexon were synthesized by Proimmune, Oxford, UK or AltaBioscience, University of Birmingham, Edgbaston, Birmingham, UK.Lyophilized peptides were reconstituted at 5 mg/ml in DMSO.

ii. PBMC Stimulation

15×10⁶ fresh/frozen PBMCs were pelleted in a 15 ml tube and pulsed for30-60 min at 37° C. with peptide libraries/pepmixes, either singly orpooled, at a concentration of 100 ng/peptide/15×10⁶ PBMCs. Afterincubation cells were resuspended in CTL media alone or supplementedwith cytokines (as outlined below) and transferred to a G-Rex10 (WilsonWolf Manufacturing Corporation, New Brighton, Minn.) (15×10⁶/G-Rex10) orplated out in a 24-well plate (2×106/well). Media and cytokines werereplenished on day 5, and cultures were split when they reached adensity>50×10⁶/G-Rex10 or >3×10⁶ cell/24-well. On day 9-12, CTLs wereharvested, counted and used for phenotypic and functional studies.

iii. Cytokines for Promoting CTL Activation and Expansion

The inventors compared 4 conditions; (i) no cytokine, (ii) IL7 (10ng/ml)+IL4 (1666 U/ml), (iii) IL15 (5 ng/ml) (R&D Systems, Minneapolis,Minn.) and (iv) IL2 (20 U/ml). Cytokines were added to CTLs at day 0 andreplenished on day 5. In some embodiments, 400 U of IL4 is employed.

iv. CTL Expansion

For expansion CTLs were restimulated at a S:R ratio of 1:1 withirradiated (30Gy) pepmix-pulsed autologous PHA blasts in CTL media withIL4+7 and IL15 (5 ng/ml) on the day of restimulation and fed with IL15twice weekly. Seven days later CTLs were harvested, and used for furtherstudies.

C. Flow Cytometry

i. Immunophenotyping

CTLs were surface-stained with monoclonal antibodies to: CD3, CD4, CD8,CD16, CD56, CD28, CD45RO, and CD62L (Becton Dickinson BD, FranklinLakes, N.J.). Cells were washed once with phosphate-buffered saline(PBS) (Sigma, St Louis, Mo.) containing 2% FBS (HyClone, Thermo FisherScientific Inc, NH), pelleted, and antibodies added in saturatingamounts (10 μl). After 15 min at 4° C. in the dark, cells were washedtwice and analyzed. Approximately 20,000 live cells were acquired usinga FACSCalibur equipped with Cell Quest software

ii. CFSE

To measure cell proliferation PBMCs were isolated, pelleted and pulsedwith pp65 pepmix (100 ng/15×10⁶ PBMC) for 30-60 min. Next PBMCs werewashed twice using PBS+0.1% FBS and incubated for 10 min with 150l/20×10⁶ PBMC 10 μM. CSFE. Subsequently FBS was added at a 1:1 ratio andincubated for 10 min at 37° C. After CFSE labeling PBMCs were washedtwice using PBS+2% FBS and plated at a concentration of 1×10⁶/ml in CTLmedia with cytokines. Dilution of CFSE was examined every 2-3 days byflow after surface staining with CD3, CD4, CD8 and CD56.

iii. FoxP3 Staining

To measure regulatory T cells Foxp3 staining was performed using thee-Bioscience FoxP3 staining kit. Briefly, CTLs were rested in CTL mediafor 48 h, then 1×10⁶ CTLs were resuspended in PBS+2% FBS and surfacestained for CD3, CD25 and CD4. After washing the cells were resuspendedin 1 ml Fixation/Permeabilizastion solution and incubated for 1 h at 4°C., then washed, resuspended in permeabilization buffer and incubatedwith 0.2 μl isotype or 10 μl FoxP3 antibody (Clone PCH101) for 30 min at4° C. After a final wash cells were acquired using a FACSCaliburequipped with Cell Quest software.

iv. Intracellular Cytokine Staining

CTLs were harvested, resuspended at a concentration of 5×10⁶/ml in CTLmedia and plated at 200 μl/well in a 96 well plate. The cells were thenstimulated with 100 ng of test or control pepmix in the presence ofBrefeldin A (1 μg/ml), (BD) CD28 and CD49d (1 μg/ml) for 5-7 hours.Subsequently, CTLs were washed with PBS+2% FBS, pelleted, and surfacestained with CD8, CD4 and CD3 (10 μl/antibody/tube). After 15 mins,cells were washed twice, pelleted, fixed and permeabilized withCytofix/Cytoperm solution (BD) for 20 mins at 4° C. in the dark. Afterwashing twice with PBS/2% FBS containing 0.1% saponin (Calbiochem, EMDChemicals, NJ) cells were incubated with 20 μl IFNγ and/or TNFαantibodies (BD) for 30 min at 4° C. in the dark. Cells were then washedtwice with cold PBS/2% FBS containing 0.1% saponin and at least 200,000live cells from each population were analyzed with a FACSCaliburequipped with Cell Quest software (BD).

D. Functional Studies

i. Multiplex Assay

To assess cytokine production the inventors used a multiplex assays.1×105 pp65-CTLs were restimulated using 500 ng/ml pp65 or controlpepmix. After 16 hrs supernatant was collected and the cytokine profileassessed using the MILLIPLEX High Sensitivity Human Cytokine MagneticBead Panel (Millipore, Billerica, Mass.). Specifically, 50 μlsupernatant was incubated overnight at 4° C. with cytokine antibodybeads. After incubation, samples were washed and incubated for 1 hr atroom temperature (RT) with the biotinylated detection antibody. FinallyStreptavidin-Phycoerythrin was added for 30 min at RT, then samples werewashed and analyzed using the Luminex 200 instrument. Samples were runin duplicate.

ii. Enzyme-Linked Immunospot Assay

The inventors used ELISpot to quantify IFNγ-producing T cells and assessthe breadth of reactivity in the CTL lines. The populations wereserially diluted from 4-1×10⁵ cells/well, and antigen-specific activitymeasured after direct pepmix or peptide mini-pool stimulation. Eachcondition was run in triplicate. After 20 hours, plates were developedas previously described ²², dried overnight at RT, then sent to ZellnetConsulting, New York, N.Y. for quantification. SFC and input cellnumbers were plotted, and a linear regression calculated after excludingplateau data points.

iii. TCR Avidity Assessment

TCR avidity was assessed by IFN ELIspot. 2×10⁵ CTLs were stimulated withserial dilutions of pepmixes (pp65, Hexon) or 9 mer peptides (NLV-pp65:NLVPMVATV HLA-A2 restricted, QYD-pp65: QYDPVAALF HLA-A24 restricted;TDL-Hexon: TDLGQNLLY HLA-A1 restricted). The frequency of T cellsspecific for each antigen/peptide was expressed as a percentage of themaximal SFC/input cell number.

iv. Chromium Release Assay

The inventors measured the cytotoxic specificity in a standard 4 hr Cr⁵¹release assay, using E:T ratios of 40:1, 20:1, 10:1, and 5:1. CTLs wereused as effectors and the targets were PHA blasts pulsed with pepmixes.Autologous and allogeneic PHA blasts alone or loaded with an irrelevantpepmix were used as specificity and alloreactivity controls. Thepercentage of specific lysis was calculated as [(experimentalrelease—spontaneous release)/(maximum release—spontaneous release)]×100.

REFERENCES

All patents and publications mentioned in this specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications herein are incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by referencein their entirety.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of generating cytotoxic T-lymphocytes(CTLs) that target at least one antigen from two or more viruses,comprising the steps of: contacting a plurality of peripheral bloodmononuclear cells with at least two libraries of peptides, saidlibraries of peptides each comprising peptides that correspond to aparticular viral antigen; and expanding the plurality of cells in thepresence of one or more cytokines.
 2. The method of claim 1, whereinsaid method occurs in the absence of exposing the libraries to isolatedpeptide-pulsed dendritic cells prior to expanding the CTLs.
 3. Themethod of claim 1, wherein the one or more cytokines are selected fromthe group consisting of IL4, IL7 and a combination thereof.
 4. Themethod of claim, wherein the peptides are further defined as peptidesthat overlap in sequence to span part or all of a viral antigen.
 5. Themethod of claim 3, wherein the peptides overlap by at least three aminoacids.
 6. The method of claim 3, wherein the peptides are at least sevenamino acids in length.
 7. The method of claim 1, wherein the viruses areselected from the group consisting of EBV, CMV, Adenovirus, BK virus,HHV6, RSV, Influenza, Parainfluenza, Bocavirus, Coronavirus, LCMV,Mumps, Measles, Metapneumovirus, Parvovirus B, Rotavirus, West NileVirus, JC, HHV7, and a combination thereof.
 8. The method of claim 1,wherein the virus is EBV and the antigen is selected from the groupconsisting of EBNA1, LMP2, and BZLF1.
 9. The method of claim 1, whereinthe virus is CMV and the antigen is selected from the group consistingof IE1 and pp65.
 10. The method of claim 1, wherein the virus is Adv andthe antigen is selected from the group consisting of Hexon and penton.11. The method of claim 1, wherein the virus is BK virus and the antigenis selected from the group consisting of LT and VP-1.
 12. The method ofclaim 1, wherein the virus is HHV6 and the antigen is selected from thegroup consisting of U14, U11, U71, U54, and U90.
 13. The method of claim1, wherein the virus is RSV and the antigen is selected from the groupconsisting of N and F.
 14. The method of claim 1, wherein the virus isInfluenza and the antigen is selected from the group consisting of MP1and NP1.
 15. The method of claim 1, wherein the CTLs are administered toan individual.
 16. The method of claim 1, wherein the CTLs areadministered to an immunocompromised individual.
 17. The method of claim15, wherein the individual has had allogeneic stem cell transplant. 18.The method of claim 14, wherein the cells are administered by injection.19. The method of claim 17, wherein the injection is intravenous. 20.The method of claim 1, wherein the CTLs are further defined aspolyclonal CD4+ and CD8+ CTLs.
 21. The method of claim 14, wherein thePBMCs are allogeneic to the individual.
 22. The method of claim 14,wherein the PBMCs are autologous to the individual.
 23. The method ofclaim 1, further comprising the step of exposing the CTLs to one or morecompositions that stimulate cell division.